Interferons are Glycoprotein messengers produced by the host cells of the body in response to the infections. Interferon secretion induces the protective defense mechanisms of the immune system like activation of Natural killer cells and Macrophages there by starting a screening mechanism to identify the infection or tumor cells by inducing antigen representation to T-Lymphocytes.
Interferon alpha-2a proteins are produced by leukocytes. They mainly up regulate the expression of MHC I proteins and allow increase in presentation of peptides derived from viral antigens. This enhances the activation of CD8+ T cells that are the precursors for cytotoxic T lymphocytes (CTLs) and makes the macrophage a better target for CTL-mediated killing. Interferon alpha also induce the synthesis of several key antiviral mediators, including 2′-5′ oligoadenylate synthetase (2′-5′ A synthetase) and protein kinase R.
This mechanism of Interferon alpha makes it useful for treatment of various diseases like chronic hepatitis C and hairy cell leukemia, chronic phase Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) patients.
Interferon alpha-2b is indicated for the treatment of chronic hepatitis C in patients 3 years of age and older with compensated liver disease.
Interferon alpha-2a is approved for the treatment of chronic hepatitis C and hairy cell leukemia in patients 18 years of age or older.
Production of Recombinant Interferon alpha is done using E. coli as a host. The cDNA coding for Interferon is cloned into vector and the vector is transformed into E. coli to produce the recombinant protein. The protein produced using the gene is low in quantity because of the codon bias of the E. coli towards the genes of the Human origin. Codon bias is due to the differences in the codon usage in the bacterial genome and the human genome. The codons present rarely in the genome of E. coli are bound to have lesser amount of t-RNA and the scarcity of the t-RNA can cause the premature termination of the m-RNA. This will result in inadequate use of the cellular mechanism for production of the mRNA. Low mRNA yield will affect the protein production. Hence there is need to modify the codons of the gene coding for protein to facilitate proper utilization of available tRNA thereby increasing the production of mRNA coding the protein of interest. Increased amount of mRNA will improve the protein production levels.
The codon optimization is done by substituting the rare codons with abundant codons. But sometimes such substitution may result into production of high GC content regions, complementary regions, or internal ribosome entry sites or poly adenylation sites. This can affect the mRNA formation by causing formation of improper mRNA or causing early termination of mRNA. Thus it is necessary to consider various factors which can affect mRNA stability and cause decrease in the expression levels of the mRNA coding for the protein of interest. As the number of changes in the native cDNA increase the chances of introduction of unfavorable regions may increase. The ideal codon optimization strategy involves identifying the locations where the codons can be substituted to improve the yield of expression. The strategy should be applied carefully to avoid substitution of rare codons at certain location where such substitution can negatively impact the mRNA stability and level of protein expression.
For this reason, there is need to produce the codon optimized cDNA sequences coding for Interferon alpha-2a gene and increasing the expression of the protein.